ANKRD1 is a mesenchymal-specific driver of cancer-associated fibroblast activation bridging androgen receptor loss to AP-1 activation

There are significant commonalities among several pathologies involving fibroblasts, ranging from auto-immune diseases to fibrosis and cancer. Early steps in cancer development and progression are closely linked to fibroblast senescence and transformation into tumor-promoting cancer-associated fibroblasts (CAFs), suppressed by the androgen receptor (AR). Here, we identify ANKRD1 as a mesenchymal-specific transcriptional coregulator under direct AR negative control in human dermal fibroblasts (HDFs) and a key driver of CAF conversion, independent of cellular senescence. ANKRD1 expression in CAFs is associated with poor survival in HNSCC, lung, and cervical SCC patients, and controls a specific gene expression program of myofibroblast CAFs (my-CAFs). ANKRD1 binds to the regulatory region of my-CAF effector genes in concert with AP-1 transcription factors, and promotes c-JUN and FOS association. Targeting ANKRD1 disrupts AP-1 complex formation, reverses CAF activation, and blocks the pro-tumorigenic properties of CAFs in an orthotopic skin cancer model. ANKRD1 thus represents a target for fibroblast-directed therapy in cancer and potentially beyond.

For all statistical analyses, confirm that the following items are present in in the figure legend, table legend, main text, or or Methods section.

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as as a discrete number and unit of of measurement A statement on on whether measurements were taken from distinct samples or or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.

A description of of all covariates tested
A description of of any assumptions or or corrections, such as as tests of of normality and adjustment for multiple comparisons A full description of of the statistical parameters including central tendency (e.g.means) or or other basic estimates (e.g.regression coefficient) AND variation (e.g. standard deviation) or or associated estimates of of uncertainty (e.g.confidence intervals) For null hypothesis testing, the test statistic (e.g.F, t, r) with confidence intervals, effect sizes, degrees of of freedom and P value noted Give P values as exact values whenever suitable.
For Bayesian analysis, information on on the choice of of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of of the appropriate level for tests and full reporting of of outcomes Estimates of of effect sizes (e.g.Cohen's d, Pearson's r), ), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.In all the experiments, the animals were injected contralaterally with both control and treated cells.In the gavage experiments the animals were randomly assigned to control or treated group.

Materials
The researchers involved in the study were not blinded during sample obtainment or data analysis.Considering the multiple aspects of the study, complete blinding of the investigators was not possible for the data collection, nor relevant considering the cross-check of the results.
In vitro experiments and bioinformatic data analysis were cross checked by the Investigators involved in the study.The in vivo experiments were carried out as a team.
A full list of primary and secondary antibodies, including catalog numbers, clone names and dilutions, used in the study is provided in the Supplementary Data file 8.
used in in the manuscript was obtained from publicly available depositories and websites, including GEO and MSigDB.The GEO numbers are specified in in Methods section of of the manuscript.Light Cycler 480 (Roche) associated software, ZEISS LSM880 confocal microscope, and NanoZoomer 560 microscope associated softwares Nikon Ndp.View2.Fiji/lmageJ.v1.0.0 R package EPIC v1.1.5R package survival v2.43-3 Enrichr Graph Pad Prism Software IGV Software Microsoft Excel 365 PeaksAnnotator (HOMER) Bowtie2 Version 2.3.0MACS the Burrows-Aligner (BWA) 70 70 PeaksAnnotator (HOMER) Bowtie2 Version 2.3.0 nature portfolio | reporting summary April 2023 Randomization Blinding Reporting for specific materials, systems and methods and strains are reported in the figure legends for each experiment).All attempts were successful.No cell line or data were excluded.